Low Fragmented Chromatin Concentration (ChIP (CST Guide))

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Accepted Answer

DNA concentration of fragmented chromatin preparation is below expected ranges (e.g., <100 µg/ml for HeLa cells, <20 µg/ml for brain tissue). Insufficient material for recommended 5-10 µg chromatin per IP reaction. Common causes: 1) Insufficient starting material: fewer cells or less tissue than protocol specification (e.g., <4×10⁶ HeLa cells or <25 mg tissue) 2) Incomplete cell or tissue lysis during nuclear extraction step 3) Enzymatic protocol: incomplete nuclear membrane disruption after micrococcal nuclease digestion 4) Poor tissue disaggregation (especially brain tissue without Dounce homogenizer) Solutions: 1) Count cells accurately before crosslinking or weigh tissue precisely to ensure adequate starting material 2) If concentration is near 50 µg/ml, add additional chromatin volume to reach 5-10 µg per IP and proceed 3) Enzymatic protocol: visualize nuclei under microscope before and after sonication to confirm complete lysis 4) Use Dounce homogenizer for brain tissue disaggregation; Medimachine inadequate for this tissue type 5) Harvest more than 25 mg tissue per IP for low-yield tissue types (brain, heart: 2-5 µg per 25 mg)